mRNA Expression Of mTOR Research Articles

Screening and biological evaluation of myricetin as a multiple target inhibitor insulin, epidermal growth factor, and androgen receptor; in silico and in vitro.

Myricetin is a naturally omnipresent benzo-α-pyrone flavonoids derivative; has potent anticancer activity. Receptor tyrosine kinases family provides the decisive role in cancer initiation and progression. These receptors have recently caught the attention of the researchers as an attractive target to combat cancer, owing to the evidences endorsed their over-expression on cancer cells. This study is a concerted effort to explore the potent and specific multi-targeted inhibitor against RTKs and AR\ER employing molecular docking approach. IR, IGF1R, EGFR, VEGFR1, VEGFR2, and AR\ER were chosen as a protein and natural compounds as a ligand. Molecular docking procedure followed by using Maestro 9.6 (Schrödinger Inc). All natural compounds were docked with the X-ray crystal structures of selected proteins by employing grid-based ligand docking with energetics Maestro 9.6. IBS natural compounds docked with each selected protein molecules by using GLIDE high throughput virtual screening. On the basis of Gscore, we selected 20 compounds from IBS (50,000 compounds) along with 68 anticancer compounds from published literature for GLIDE extra precision molecular docking. Calculated docking free energy yielded the excellent dock score for the myricetin when docked with proteins EGFR, IR, and AR\ER. Protein-ligand interactions profile highlighted that the lipophilic, hydrogen bonding and π-π stacking interactions play a central role in protein-ligand interactions at the active site. The results of MTT assay reveal that the myricetin inhibit the viability and proliferation of cancer cells in a dose-dependent manner. Treatment with the myricetin led to down-regulation of mRNA expression of EGFR, IR, mTOR, and Bcl-2. Although, further in vitro and in vivo experimental studies are required for the experimental validation of our findings.

Suppression of lentiviral vector-mediated mTOR gene silencing on growth of human lens epithelial cell in vitro

Background Mammalian target of raparrmycin (mTOR) acts as a critical molecular link between growth signals and the processes underlying cell growth. The overexpression of mTOR in human lens epithelial cells (HLECs) is associated with posterior capsular opacification (PCO). Applying the mTOR shRNA transfection technique to silence mTOR in HLECs is significant for the targeting treatment of PCO. Objective This study was to investigate the function and morphology of HLECs infected by mTOR shRNA lentiviral vector. Methods 293T cells were transfected by recombinant plasmid containing mTOR shRNA, blank plasmid and PBS to prepare different lentiviral vectors. HLECs were routinely cultured and were divided into the mTOR shRNA transfected 1, 2, 3 groups, blank plasmid group and PBS group, and different lentiviral vectors were added in the medium to infect the cells. The expression of mTOR protein in HLECs was detected using Western blot to determine mTOR gene silence, and the group with the highest efficiency of silence was screened as the sequent experiment group. The proportion of HLECs in the G1 phase was analyzed by flow cotymetry. The expressions of α-smooth muscle actin (α-SMA) and E-Cadherin proteins in the cells were assayed by Western blot. The viability of HLECs was assessed by cell counting kit-8 (CCK8) test. Results Successfully packaged lentivirus were found in the supernatant after infection of mTOR shRNA in the mTOR shRNA transfected 1, 2, 3 groups with the green fluorescence in >80% cells, while green fluorescence protein (GFP) response was absent in the blank plasmid group and the PBS group. The HLECs of mTOR shRNA transfected groups and blank plasmid group grew well due to the resistance for puromycin of plasmid, however, more dead cells were found in the PBS group. Western blot revealed the weakest expression of mTOR mRNA in the cells in the mTOR shRNA transfected 3 group. The expressing bland of α-SMA in the HLECs was weaker in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group, and the expression of E-Cadherin was stronger in in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group. The absorbance of the mTOR shRNA transfectd 3 group was significantly declined in comparison with the blank plasmid group and the PBS group, with significant difference among the three groups in both 1 hour and 2 hours after experiment (all at P<0.05). The proportion of cells in G1 phase was (60.00±1.78)% in the mTOR shRNA transfected 3 group, which was significantly increased in comparison with (53.48±1.86)% in the blank plasmid group and (53.02±1.49)% in the PBS group (F=18.910, P=0.002). Conclusions mTOR gene is silenced after mTOR shRNA plasmid transfected into HLECs. This procedure can invert the epithelial-mesenchymal transition (EMT), inhibit cell growth and put off cellular cycle in HLECs in vitro. Key words: Lentiviral vector; Short hairpin RNA; Posterior capsule opacification; Mammalian target of raparrmycin; Epithelial-mesenchymal transition; Lens, crystal; Epithelial cells; Gene silence

Decreased AKT1/mTOR pathway mRNA expression in short-term bipolar disorder

Strong evidence implicates intracellular signaling cascades dysfunction in the pathophysiology of Bipolar Disorder (BD). Regulation of AKT/mTOR pathway is a critical signaling pathway in synaptic neurotransmission and plasticity, also modulating cell proliferation and migration.Gene expression of the AKT/mTOR pathway was assessed in 25 BD (DSM-IV-TR criteria) unmedicated depressed individuals at baseline and after 6 weeks of lithium therapy and 31 matched healthy controls. Decreases in blood AKT1 and mTOR mRNA expression, as well as in BAD/BCL-2 expression ratio were observed in short-term BD patients during depressive episodes in comparison to healthy controls. There was no significant change in the expression of AKT1, mTOR, BCL-2, BAD and NDUFA6 after lithium therapy in the total group of BD subjects. However, the changes in AKT1 expression after lithium treatment were positively correlated with depression improvement. An integrated activity within this pathway was observed at both baseline and post-treatment. The present results support an integrated AKT/mTOR signaling pathway activity in a similar fashion to the described in previous human postmortem and rodents brain studies. Overall, the results reinforce a role for AKT1 and mTOR in the pathophysiology of BD and support the relevance of blood mRNA expression as a valid surrogate biological source to study brain intracellular signaling cascades changes and convergent molecular pathways in psychiatric disorders.

Effect of Nourishing "Yin" Removing "Fire" Chinese Herbal Mixture on Hypothalamic Mammalian Target of Rapamycin Expression during Onset of Puberty in Female Rats.

Aim. The present study aims to investigate the effects of nourishing “Yin” removing “Fire” (NYRF) Chinese herbal mixture on puberty onset and hypothalamic mTOR expression in female rats. Materials and Methods. Forty female 20-day-old Sprague-Dawley rats were randomly divided into Chinese herbal mixture (CHM) and normal saline (NS) groups. Rats in CHM and NS were treated with NYRF mixture and normal saline, respectively, from d22. Rats in each group were sacrificed on d28, d31, and d34. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), and estradiol (E2) levels were analyzed by ELISA. Hypothalamic mTOR mRNA expression levels were determined by RT-PCR and hypothalamic p-mTOR protein levels were assayed by western blot. Results. The vaginal opening time in CHM group was significantly delayed (P < 0.05). On d31, in comparison with NS group, the coefficients of uteri and ovaries, levels of serum LH and E2, and the expression levels of hypothalamic mTOR mRNA and p-mTOR protein were significantly lower in CHM group (P < 0.05). Conclusion. The mechanism by which the nourishing “Yin” removing “Fire” Chinese herbal mixture delays puberty onset may be associated with the inhibition of the hypothalamic mTOR signaling.

Effect of Mechanism of Action of Different ω-6/ω-3 Polyunsaturated Fatty Acids Ratio on the Growth of Endometrial Carcinoma Mice.

To explore the effect and mechanism of action of different ω-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio on the expression of AKT and mTOR in mice bearing endometrial carcinoma. Once the human endometrial carcinoma xenograft models were successfully established, 40 BALB/C mice were randomized into five groups: group A (ω-6 PUFAs), group B (10:1 ω-6/ω-3 PUFAs), group C (control group), group D (1:1 ω-6/ω-3 PUFAs), and group E (ω-3 PUFAs). Six weeks post-treatment, mice were sacrificed and the xenograft tissues were harvested for immunohistochemical SP analysis of AKT and mTOR expression. AKT and mTOR mRNA expression was determined by reverse transcription polymerase chain reaction. Group A and group B had the highest positive expression of AKT and mTOR, with increased mRNA expression. Group D and group E had the lowest positive expression of AKT and mTOR, with decreased mRNA expression. There was a positive correlation between the expression of AKT and that of mTOR (r=0.92). Thus, ω-6/ω-3 PUFAs in different proportions are associated with the mRNA expression of AKT and mTOR in the tissues of mouse xenograft model of human endometrial cancer.

Primordial follicle activation in the ovary of Ames dwarf mice.

BackgroundThe insulin receptor substrate 1 (IRS1), phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt1), Forkhead Box O3a (FOXO3a) pathway is directly involved in aging and ovarian activation of follicle growth. Therefore, the aim of this work was to measure the expression of genes related to the ovarian pathway for activation of primordial follicles and FOXO3a protein phosphorylation between young and old female Ames dwarf (df/df) and normal (N) mice.MethodsFor this study ovaries from N (n = 10) and df/df (n = 10) female mice were collected at 5–6 months of age and at 21–22 months of age. For immunohistochemistry ovaries from 12 month-old and df/df mice were used.ResultsThe expression of Irs1, Pi3k, Akt1, mammalian target of rapamycin (Mtor), suppressor of cytokine signaling −2 (Socs2), Socs3 was lower (P < 0.05) in older than younger N mice and not different (P > 0.05) between young and old df/df mice. The expression of Foxo3a was also lower (P < 0.05) in old than younger N and df/df mice and was higher (P < 0.05) in old df/df than N mice. Expression of Amh was lower (P < 0.05) in old than young N and df/df mice and was higher (P = 0.0009) in df/df than N mice. Imunnostaining for p-FOXO3 was lower in df/df than N mice (P < 0.001), although FOXO3 immunostaining was not different (P > 0.05) between df/df and N mice.ConclusionsIn sum, the present study indicates that lower expression of Irs1, Socs2, Socs3, Akt1, Pi3k, Mtor and Foxo3a mRNA in the ovaries of older mice of both genotypes is associated to a reduced ovarian activity revealed by lower expression of Amh mRNA. At the same time, ovaries of old df/df mice maintained higher expression of Foxo3a mRNA, which was associated to higher ovarian activity. We have shown that df/df females have a lower level of p-FOXO3 in oocytes from primordial/primary follicles, an important activator of follicular growth. Therefore, this study strongly indicates that Prop1df mutation causes delayed ovarian aging.

The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability.

IntroductionMicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy.MethodsThe bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p < 0.05 was considered significant.ResultsAfter miR-100 transfection, there was a significant reduction in the mRNA of mTOR (p = 0.006), SMARCA5 (p = 0.007) and BAZ2A (p = 0.029) in RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma.ConclusionmiR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use as tumor markers and targets for new therapies.

Characterization of the mTOR pathway in human normal adrenal and adrenocortical tumors.

The mTOR pathway has recently been suggested as a new potential target for therapy in adrenocortical carcinomas (ACCs). The aim of the current study is to describe the expression of the mTOR pathway in normal adrenals (NAs) and pathological adrenals and to explore whether there are correlation between the expression of these proteins and the in vitro response to sirolimus. For this purpose, the MTOR, S6K1 (RPS6KB1), and 4EBP1 (EIF4EBP1) mRNA expression were evaluated in ten NAs, ten adrenal hyperplasias (AHs), 17 adrenocortical adenomas (ACAs), and 17 ACCs by qPCR, whereas total(t)/phospho(p)-MTOR, t/p-S6K, and t/p-4EBP1 protein expression were assessed in three NAs, three AHs, six ACAs, and 20 ACCs by immunohistochemistry. The effects of sirolimus on cell survival and/or cortisol secretion in 12 human primary cultures of adrenocortical tumors (ATs) were also evaluated. In NAs and AHs, layer-specific expression of evaluated proteins was observed. S6K1 mRNA levels were lower in ACCs compared with NAs, AHs, and ACAs (P<0.01). A subset of ATs presented a moderate to high staining of the evaluated proteins. Median t-S6K1 protein expression in ACCs was lower than that in ACAs (P<0.01). Moderate to high staining of p-S6K1 and/or p-4EBP1 was observed in most ATs. A subset of ACCs not having moderate to high staining had a higher Weiss score than others (P<0.029). In primary AT cultures, sirolimus significantly reduced cell survival or cortisol secretion only in sporadic cases. In conclusion, these data suggest the presence of an activated mTOR pathway in a subset of ATs and a possible response to sirolimus only in certain ACC cases.

Effect of BIM and mTOR expression on clinical outcome to erlotinib in EGFR-mutant non-small cell lung cancer (NSCLC) patients (p).

8072 Background: Sixty percent of EGFR-mutant NSCLC p respond to erlotinib, but overall survival (OS) is the same for upfront chemotherapy. Priming BIM, a pro-apoptotic signaling BH3-only protein, induced sensitivity to erlotinib in EGFR-mutant cell lines (Costa el at. PLoS Med 2007). BIM was related to response and progression-free survival (PFS) in clinical tumor samples, but OS was not reported (Faber et al. Cancer Disc 2011). Mammalian target of rapamycin (mTOR) negatively regulates apoptosis and could influence response to erlotinib. Methods: We assessed static levels of total BIM, BIM-extra long (BIM-EL – a main isoform of BIM), and mTOR mRNA expression and correlated findings with response and OS in 57 erlotinib- or chemotherapy-treated EGFR-mutant NSCLC p in the EURTAC trial. Results: Median age 65; 70.2% female; 59.6% never-smokers; 91.2% adenocarcinoma. Response rate 88.9% for erlotinib-treated p with high total BIM levels, compared to 22.2% with low/intermediate total BIM levels (P=0.0027). Sen...

P0217 Potentially functional polymorphisms in the mTOR gene and gastric adenocarcinoma susceptibility in an eastern Chinese population

Background mTOR is an important intracellular mediator involved in the PI3K/AKT signalling pathway and a critical regulator of cell growth and proliferation in response to environmental and nutritional conditions. We evaluated potentially functional polymorphisms in the 5′ untranslated region (UTR) of the mTOR gene associated with the risk of gastric cancer. Methods In this case–control study with 1123 patients and 1113 healthy controls matched for age and gender, we genotyped four potentially functional single nucleotide polymorphisms (SNPs) located in 5′ UTR of the mTOR gene and evaluated their association with risk of gastric cancer. Findings In individual polymorphism analysis, the rs1034528 G > C was associated with 1.21-times increased risk of gastric cancer under a dominant model (adjusted odds ratio 1.21, 95% confidence interval [CI] 1.01–1.46). When the four polymorphisms were combined, the risk of gastric cancer increased in a dose-dependent manner as the number of risk genotypes increased (ptrend = 0.001). Patients with three or four risk genotypes had a significantly higher risk of gastric cancer than those with 0–2 risk genotypes (adjusted odds ratio 1.41, 95% CI 1.15–1.73). Stratification analysis indicated that the combined effect of risk genotypes was more evident in subgroups of older age (>59 years), men, smokers, non-drinkers, patients with gastric cardia adenocarcinoma, and those with clinical stage III and IV disease. Further analysis using mRNA expression data from the HapMap project suggested that both CG and CC carriers of rs1034528 G > C were associated with non-significantly decreased expression of mTOR mRNA in normal cell lines from 45 Chinese patients (p = 0.97 for CG carriers, p = 0.38 for CC carriers). Interpretation Functional mTOR variants may contribute to the risk of gastric cancer. Larger studies with different ethnic populations are warranted to validate these findings.

Rapamycin rescues the poor developmental capacity of aged porcine oocytes.

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68) or control oocytes (44 h IVM; 42.14±4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector

The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.

Characterization of the endocrine, digestive and morphological adjustments of the intestine in response to food deprivation and torpor in cunner, Tautogolabrus adspersus

The cunner, Tautogolabrus adspersus, is a marine teleost endemic to the cold waters of the Northwest Atlantic Ocean. The cunner is non-migratory and is known for its remarkable ability to endure the freezing winter months with little to no food by entering a torpid/dormant state. To evaluate the physiological strategies employed by the cunner's intestinal tract to withstand food deprivation, fish were sampled for their gut after a four-week period of acute food deprivation during their summer (active/feeding) state, as well as after 4months of overwinter fasting. Digestive capacity was evaluated by measuring digestive enzyme activity and related mRNA transcript expression for trypsin, alkaline phosphatase, alanine aminopeptidase and lipase. In order to assess how gut hormones affect/are affected by acute fasting and torpor, we examined the intestinal mRNA expression of several putative appetite regulators, i.e. CCK, apelin, orexin and mTOR. Short-term summer fasting induced a reduction in the activity, but not the transcript expression, of all digestive enzymes examined as well as a reduction in gut apelin mRNA. Torpor induced a reduction in the activity of all enzymes with the exception of alanine aminopeptidase, and a decrease in mRNA levels of alanine aminopeptidase, orexin, CCK and mTOR. Our results suggest that both acute fasting and long-term fasting induce a reduction in the intestinal function of cunner, as evidenced by an overall decrease in the activities of digestive enzymes and mRNA expression of several factors involved in feeding and digestion.

Activation of mammalian target of rapamycin signaling in skeletal muscle of neonatal chicks: Effects of dietary leucine and age

The mammalian target of rapamycin (mTOR) signaling pathway is necessary for cellular protein synthesis regulation. Leucine was reported to stimulate muscle protein synthesis in mammalian embryos and neonates, but in higher animals (chickens) the effect of dietary leucine on mTOR signaling is unknown. Thus, we investigated the effects of dietary leucine and age on mRNA expression and phosphorylation of mTOR as well as its downstream targets, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in chick pectoral muscles. One hundred eighty newly hatched male chicks were randomly assigned to 1 of 3 dietary leucine treatment groups (1.43, 1.73, and 2.03% leucine) for 14 d, respectively. Each treatment group consisted of 6 cages with 10 chicks each. On d 3, 7, and 14, plasma insulin and leucine were measured and target gene expression and phosphorylation was assessed. Dietary leucine influenced plasma leucine but not insulin, and plasma leucine and insulin declined with chick age. The mTOR, S6K1, and 4E-BP1 mRNA expression and phosphorylation within chick pectoral muscles were upregulated with increased dietary leucine but downregulated with increased chick age. Thus, high dietary leucine activates target of rapamycin signaling pathways in skeletal muscle of neonatal chicks to stimulate muscle protein synthesis, and this pathway is attenuated with aging.

The Mammalian Target of Rapamycin Signalling Pathway Is Involved in Osteoblastic Differentiation of Vascular Smooth Muscle Cells

BackgroundVascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated. MethodsCalcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors. ResultsThe model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner. ConclusionsmTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.

Human Umbilical Cord Blood Mononuclear Cells in a Double-Hit Model of Bronchopulmonary Dysplasia in Neonatal Mice

BackgroundBronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia.ObjectivesWe investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD.MethodsFor the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups.ResultsCompared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased.ConclusionThe results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.

Construction of cytoskeleton modulated by phosphatase and tensin homolog deleted on chromosome ten/mammalian target of rapamycin during the differentiation of bone marrow stem cell-derived nerve cells

Objective To observe the construction of cytoskeleton modulated by phosphatase and tensin homolog deleted on chromosome ten (PTEN)/mammalian target of rapamycin (mTOR) during the nerve cell differentiation by human bone marrow stem cells in vitro,and to analyze the implication.Methods Adult bone marrow cells were isolated and induced into nerve-like cells by some cytokines in vitro after 2 weeks.The mRNA expression of mTOR which is the downstream molecule of PTEN was compared between un-treated and treated by the BPV (a type of PTEN inhibitor) using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).The construction patterns of cytoskeleton were indicated by Phalloidin-fluoresceine isothiocyanate (FITC),and observed by the fluorescence microscopy.Results Neuron specific enolase (NSE) and β-Ⅲ tubulin were expressed in these cells after the induction by adult human bone marrow stem cells.nestin,the marker of neural progenitors was expressed increasingly at the beginning,and decreasingly subsequently.The mRNA expression level of mTOR was increased and the lenth of the neurites was from (4.58 ± 1.21) μm to (9.09 ±2.68) μm after the cells were treated by BPV (P ＜ 0.05).Conclusion When the nerve cells derived from bone marrow stem cells obtain the mature differentiation,PTEN/mTOR may possess the the ability of cytoskeleton construction modulation in the neuritis of nerve cells in vitro. Key words: Nerve cell; Cell differentiation; Phosphatase and tensin homolog deleted in chromosome 10; Mammalian target of rapamycin; Cytoskeleton

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

MicroRNA 100: a context dependent miRNA in prostate cancer

OBJECTIVE:MicroRNAs are noncoding RNA molecules involved in the development and progression of tumors. We have found that miRNA-100 is underexpressed in metastatic prostate cancer compared to localized disease. Conversely higher levels of miR-100 are related to biochemical recurrence after surgery. This suggests that miR-100 may be a context-dependent miRNA, acting as oncogene or tumor suppressor miRNA. Our aim is to demonstrate the role of miR-100 in the control of predicted target genes in prostate cancer cell lines.METHODS:Cell lines DU145 and PC3 were transfected with miR-100, antimiR-100 and after 24 h and 48 h of exposure, qRT-PCR and western blot were performed for mTOR, FGFR3, THAP2, SMARCA5 and BAZ2A.RESULTS:There was reduction in mTOR (p = 0.025), THAP2 (p = 0.038), SMARCA5 (p = 0.001) and BAZ2A (p = 0.006) mRNA expression in DU145 cells after exposure to miR-100. In PC3 cells, mTOR expression was decreased by miR-100 (p = 0.01). There was a reduction in the expression levels of proteins encoded by studied genes, ranging from 34% to 69%.CONCLUSIONS:We demonstrate that miR-100 is a context-dependent miRNA controlling BAZ2, mTOR, FGFR3, SMARCA5 and THAP2 that might be involved in PC progression. The elucidation of the roles of miRNAs in tumors is important because they can be used as therapeutic targets in the future.

Impaired hypertrophy in myoblasts is improved with testosterone administration

We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have a reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7,26]). Cells were exposed to low serum conditions in the presence or absence of T (100nM)±PI3K inhibitor (LY294002) for 72h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (μm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32±2.56μm to 21.02±1.89μm and 14.58±2.66μm to 18.29±3.08μm (P≤0.05) respectively after 72h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P≤0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway.